1. Field of the Invention
The present invention relates to a trehalose-producing microorganism and a process for producing trehalose. It also relates to a novel trehalose synthase protein, a trehalose synthase gene, recombinant plasmids carrying said trehalose synthase gene, and transformed microorganisms with said recombinant plasmids.
2. Description of the Prior Art
Trehalose is a non-reducing disaccharide, two saccharides of which are linked by xcex1-1,1 bond: xcex1-D-glucopyranosyl-xcex1-D-glucopyranoside. It has wide application in medicines, foods, and cosmetics. However, its utilization has been greatly restricted because its production to date has been inefficient and expensive.
Japanese Laid-open Patent Nos. Hei5-91890 and Hei6-145186 disclose methods for extracting trehalose from yeasts. There are several methods for isolating trehalose from fermented microorganism cultures, such as Arthrobacter (T. Suzuki, Agric. Biol. Chem., 33(2), 1969), Nocardia (Japanese Laid-open Patent No. Sho 50-154485), Micrococcus (Japanese Laid-open Patent No. Hei6-319578), amino acid-fermenting yeast, Brevibacterium (Japanese Laid-open Patent No. Hei5-211882), and yeast (Yoshikwa, etc., Biosci. Biotech. Biochem., 1994, 58, 1226-12300). Additionally, a method for producing trehalose by using recombinant plants including bacterial genes capable of converting glucose into trehalose is described in M. Scher, Food Processing, April, 95-96, 1993. Japanese Laid-open Patent No. 83-216695 discloses a method for converting maltose into trehalose by using maltose phosphorylase and trehalose phosphorylase. However, these methods are not effective, because their procedures are complicated and their yields are low.
Several enzymatic methods have been published recently. Japanese Laid-open Patent No. Hei7-143876 and EPO 628630 A2 discloses a two-step enzymatic conversion method in which starch is converted into trehalose by maltooligosyl trehalose synthase and maltooligosyl trehalose trehalohydrolase. Japanses Laid-open Patent No. Hei7-170977 and Korean Laid-open Patent No.95-3444 disclose one-step enzymatic conversion methods in which maltose is directly converted into trehalose by trehalose synthase. However, there is still a need to increase the titer of the trehalose synthase enzyme so that production of trehalose from maltose becomes more efficient in yield and cost.
We have invested much effort over the last several years in isolating microorganisms able to convert maltose into trehalose from soil. We have successfully screened a novel strain which highly expresses trehalose and, unexpectedly, generates no byproducts, unlike all known microorganisms. Its morphological and physiological characteristics identify it as a novel Pseudomonas stutzeri strain. This strain has been designated as Pseudomonas stutzeri CJ38.
We isolated a trehalose synthase gene from chromosomes of Pseudomonas stutzeri CJ38 and determined its nucleotide sequence by cloning it into known vector pUC 18 with restriction enzyme Sau3AI. In addition, we isolated a trehalose synthase protein from Pseudomonas stutzeri CJ38 and determined its amino acid sequence using standard methods. It was found that these sequences are apparently different from the sequences of the trehalose synthase gene and all proteins known hitherto. This invention was achieved by constructing recombinant plasmnids carrying the trehalose synthase gene so that the trehalose synthase enzyme encoded in said gene can be expressed in large amounts.
The present invention provides a novel microorganism, Pseudomonas stutzeri CJ38, that produces trehalose from maltose. This strain was deposited at the Korea Culture Center of Microorganisms, Seoul, Korea, as the accession number KCCM 10150 on Feb. 12, 1999 under the Budapest Treaty. This strain is very valuable as it does not generate byproducts such as glucose when converts maltose into trehalose.
The present invention also provides SEQ ID NO: 2, which is a novel trehalose synthase protein with the following amino acid sequence:
In addition, the present invention provides SEQ ID NO: 1, which is a novel trehalose synthase gene with the following nucleotide sequence:
The present invention also provides a recombinant plasmid containing the trehalose synthase gene with the above nucleotide sequence. In a preferred embodiment, the present invention provides are combinant plasmid pCJ104 in which the 4.7 kb Sau3AI DNA fragment of the trehalose svnthase gene of the present invention is cloned into vector plasinid pUC18. This allow for the efficient and high expression of the trehalose synthase gene. In a more preferred embodiment, the present invention provides a recombinant plasmid pCJ122 in which the 2.5 kb BamHI-BglII DNA fragment of the trehalose synthase gene of the present invention is included in a vector plasmid pUC18. allowing for a higher expression of the trehalose synthase gene.
The present invention provides a transformed E. coli with a recombinant plasmid containing the trehalose synthase gene with the above nucleotide sequence. In a preferred embodiment, the present invention provides a transformed E. coli with a recombinant plasmid pCJ104, allowing for production of high levels of the trehalose synthase protein. In a more preferable embodiment, the present invention provides a transformed E. coli with the recombinant plasmid pCJ122, allowing for production of even higher levels of the trehalose synthase protein.
The present invention provides a process for producing trehalose which comprises reacting the trehalose synthase protein with the above amino acid sequence with maltose solution to obtain trehalose.
The present invention provides a process for producing trehalose which comprises crushing a transformed E. coli with a recombinant plasmid containing the trehalose synthase gene with the above nucleotide sequence and reacting the crushed cells with maltose solution to obtain trehalose. In a preferred embodiment, the present invention provides a process for producing trehalose which comprises crushing a transformed E. coli with plasmid pCJ104, centrifuging the crushed cells and reacting the resulting supernatant with maltose solution to obtain trehalose. In a more preferable embodiment, the present invention provides a process for producing trehalose which comprises crushing a transformed E. coli with plasmid pCJ122, centrifuging the crushed cells and reacting the resulting supernatant with maltose solution to obtain trehalose.